In blood, there exist various hemocytes having different shapes and functions, including erythrocytes, leukocytes, and platelets, which play important roles in maintaining homeostatis of the living body. These mature hemocytes have their own life-spans. For maintaining the hemocyte count at a constant level, hemocytes must be incessantly produced to make up for the number of hemocytes that is lost due to the expiration of their life-spans.
In the normal healthy individual, it is presumed that daily production of hemocytes reaches as much as 2×1011 erythrocytes, 1011 leukocytes, and 1 to 2×1011 platelets. Hematopoietic stem cells play central rolls in the system to produce such an enormous number of hemocytes over a long period without being exhausted. The cells have not only self-renewal capability but also multipotentiality to differentiate to various mature hemocytes including erythrocytes, granulocytes, platelets, and lympocytes. Hematopoietic stem cells (multipotential stem cells) lose their self-renewal capability as they proliferate to become hematopoietic precursor cells (committed stem cells) destined to differentiate to the specific hemocytes. Hematopoietic precursor cells then differentiate to mature peripheral hemocytes.
It has been known that a number of cytokines regulate each step of the hematopoietic system to proliferate and differentiate hematopoietic stem cells to various mature hemocytes via hematopoietic precursor cells. At least twenty kinds of the cytokines participating in the hematopoietic system have been found at present (Masami Bessho: Igaku no Ayumi 180(13): 802-806, 1997). The genes for all have been cloned, allowing their production on a large scale by genetic engineering techniques. Stem cell factor (SCF) and flk-2 ligand are the most remarkable cytokines as factors acting on mainly hematopoletic stem cells at the early stage of hematopoiesis. SCF acts on the most undifferentiated hematopoietic stem cells. In either mice or humans, it remarkably promotes the formation of colonies of blast colony-forming unit (CFU-BL), colony-forming unit-mixed (CFU-Mix), burst forming unit-erythrocyte (BFU-e), colony-forming unit-granulocyte/macrophage (CPU-GM), eosinophil colony-forming unit (CFU-Eo), and colony-forming unit-megakaryocyte (CPU-Meg); showing a synergistic effect with various cytokines such as IL-1, IL-3, II-4, IL-5, IL-6, IL-7, IL-11, G-CSF, GM-CSF, and EPO. It has been reported that SCF alone has weak colony-stimulating activity (Tsuji, K. et al., Blood 78: 1223,1991; Shioharu, M. et al., Blood 81: 1453, 1993; Kubo, T. and Nakahata, T., Int. Hematol. 58: 153, 1993). Nevertheless, SCF is thought to be the most important cytokine for in vitro amplification of hematopoietic stem cells at present.
The gene for flk-2 ligand has been just recently cloned and its biological activity has not been fully clarified. Since it exhibits synergistic actions with many cytokines as SCF does, it is expected to be an important factor for in vitro amplification of human hematopoietic stem cells.
Some of these hematopoietic factors have been clinically applied. For example, erythropoietin (EPO), which promotes the production of erythrocytes, is used for treating renal anemia, and granulocyte colony-stimulating factor (G-CSF), which promotes the production of neutrophils is used for treating neutropenia caused by cancer chemotherapy. These contribute to improved quality of life of patients. Recently, the clinical application of thrombopoietin (TPO) for treating thrombocytopenia has been studied because it promotes the production of platelets.
On the one hand, since hematopoietic stem cells are capable of reconstituting all kinds of cells in the hematopoietic system, the transplantation of hematopoietic stem cells has been widely performed for hematopoietic tumors. Recently, the transplantation of peripheral blood stem cells has rapidly become prevalent, and gathered attention as the powerful fundamental therapy for the chemotherapy-sensitive malignant tumors including the hematopoietic organ tumors. Furthermore, as a future prospect, the transplantation of hematopoietic stem cells is expected to be introduced to many cell therapy and gene therapy protocols. For that purpose, it is necessary to establish a method for amplifying hematopoietic stem cells in vitro. However, even now, human hematopoietic stem cells have been neither isolated nor clarified as to what extent they can repeat self-renewal.